ABSTRACT

This study was conducted to investigate the efficacy of the simultaneous application of near-infrared (NIR) heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in red pepper powder and to clarify the mechanisms of the lethal effect of the NIR-UV combined treatment. Also, the effect of the combination treatment on quality was determined by measuring changes in color and pungency constituents. Simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log CFU reductions in S. Typhimurium and E. coli O157:H7, respectively, which involved 1.86- and 1.31-log CFU reductions, respectively, which were attributed to the synergistic effect. Through qualitative and quantitative analyses, damage to the cell envelope was identified as the main factor contributing to the synergistic lethal effect of NIR-UV combined treatment. Color values and capsaicin and dihydrocapsaicin content of NIR-UV simultaneously treated red pepper powder were not significantly (P > 0.05) different from those of untreated samples. These results suggest that simultaneous application of NIR and UV treatment can be effectively used to control food-borne pathogens in powdered red pepper without affecting quality.

INTRODUCTION

Dried powdered spices are widely used in processed food products and many other convenience foods. Among them, red pepper (Capsicum annuum L.) is one of the most important spices used as a natural flavoring and coloring agent worldwide. However, since red peppers are of agricultural origin, they may be burdened with high levels of mesophilic bacteria (1, 2). Pathogenic bacteria such as Salmonella spp., Bacillus cereus, and Escherichia coli have been identified in spices (3, 32). Such contaminated spices can result in serious food-borne illnesses when added to foods that undergo no further processing or are eaten raw (2, 4). In 2009, a multistate outbreak of Salmonella infections in the United States was traced to ground pepper products (http://wwwn.cdc.gov/foodborneoutbreaks/). As a consequence, ground spices should be decontaminated to prevent further food spoilage and food-borne diseases.

Different physical and chemical treatments have been used to reduce the microbial loads of powdered spices. The most frequently used decontamination techniques are irradiation, fumigation with ethylene oxide, and thermal processing (superheated steam) (1, 5). However, irradiation at high doses adversely affects the aroma of red pepper powder (6). Furthermore, in certain cases where spices were irradiated in prepackaged form to prevent postpackaging contamination, undesirable compounds (e.g., 1,3-di-tert-butylbenzene) from packaging materials migrated into the spice products (7). The use of ethylene oxide is prohibited in many countries due to possible toxic residues remaining after the process (8). The application of high-temperature steam is associated with a decrease in volatile oil content, color degradation, and an increase in moisture content of the dried spices, which leads to a decreased shelf life (9).

As none of these methods has proved to be completely satisfactory, searches for safe and efficient control methods, including investigations into the use of infrared (IR) heating and UV irradiation, for decontamination of dried spices are being undertaken (10, 11, 12). IR radiation is part of the electromagnetic spectrum, with wavelengths between those of UV and microwave radiation, and is distinguished as near IR (0.76 to 2 μm), medium IR (2 to 4 μm), and far IR (4 to 1,000 μm). IR heating has advantages over convection and conduction heating, as it heats the product directly without being influenced by air around the powdered spices, and is a fast and effective thermal process (13). This rapid surface heating can be used to improve the sealing of moisture, flavor, and aroma compounds, leading to products with better sensory characteristics (14). In our previous study, the effectiveness of near-infrared heating processing for surface pasteurization of solid foods was compared to conventional convective heating (15). Staack et al. (11) examined the effect of infrared radiation at near and medium wavelengths on the microbial decontamination of paprika powder, and 1- to 2-log reductions of microbial flora were obtained at an aw of 0.8. On the other hand, UV-C irradiation at a 253.7-nm wavelength is a nonthermal method approved for use as an antimicrobial treatment of food surfaces (16). UV-C radiation has been recommended for use in combination with other preservation techniques, since the cumulative damage based on microbial DNA appears to be effective in decreasing the overall number of bacterial cells (17). Recently, some studies demonstrated that the killing effect of UV-C irradiation was accelerated by combining treatments with other antimicrobial techniques (18, 19, 20).

Hamanaka et al. (21) reported that the combination of IR heating and UV-C irradiation was found to be effective in inactivation of Rhodotorula mucilaginosa cells on fig fruits. Erdoǧdu and Ekiz (12) also reported that combined IR and UV-C treatments reduced total mesophilic aerobic bacteria on cumin seeds to acceptable levels. However, since these combination studies were limited to sequential treatments, it may be difficult to determine their efficiency on a practical industrial scale and demonstrate their synergistic effect as an innovative antimicrobial intervention. In addition, no report has elucidated the effects of simultaneous application of both technologies on the efficiency of microbial decontamination of powdered foods.

The aims of this study were to investigate the efficacy of simultaneous combination of near-infrared heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and E. coli O157:H7, in powdered red pepper and to determine the effect of the combination treatment on quality factors of red pepper powder. Also, we explored the mechanisms of inactivation.

MATERIALS AND METHODS

Bacterial strains.Three strains each of S. Typhimurium (ATCC 19585, ATCC 43971, and DT 104) and E. coli O157:H7 (ATCC 35150, ATCC 43889, and ATCC 43890) were obtained from the School of Food Science bacterial culture collection of Seoul National University (Seoul, South Korea) for this study. Stock cultures were kept frozen at −80°C in 0.7 ml of tryptic soy broth (TSB; Difco, Becton, Dickinson, Sparks, MD) and 0.3 ml of 50% glycerol. Working cultures were streaked onto tryptic soy agar (TSA; Difco), incubated at 37°C for 24 h, and stored at 4°C.

Preparation of pathogen inocula.All strains of S. Typhimurium and E. coli O157:H7 were cultured individually in 5 ml of TSB at 37°C for 24 h, followed by centrifugation (4,000 × g for 20 min at 4°C) and washing three times with buffered peptone water (BPW; Difco). The final pellets were resuspended in BPW, corresponding to ca. 107 to 108 CFU/ml. Subsequently, suspended pellets of each strain of the two pathogenic species were combined to construct mixed culture cocktails (six strains total). These cell suspensions, consisting of a final concentration of approximately 108 CFU/ml, were used in this inactivation study. To analyze the mechanism of inactivation, each final pellet of S. Typhimurium and E. coli O157:H7 was resuspended in 5 ml of phosphate-buffered saline (PBS; 0.1 M) and inoculated into a sterile glass petri dish (16 mm [height] by 90 mm [inside diameter]).

Sample preparation and inoculation.Commercially processed dried red pepper powder was purchased at a local grocery store (Seoul, South Korea). For inoculation, 6 ml of culture cocktail was applied to 250-g samples inside sterile high-density polyethylene (HDPE) bags (300 mm by 450 mm). The inoculated samples were thoroughly mixed by hand massaging for 10 min to ensure even distribution of the pathogens and dried for 1 h inside a biosafety hood (22 ± 2°C) with the fan running until the aw of the sample equaled that of a noninoculated sample (ca. 0.68). The final cell concentration was 106 to 107 CFU/25 g. Inoculated red pepper powder samples were then immediately used in each experimental batch.

Near-infrared heating and UV irradiation.A stainless chamber (concave upwards base, 380 by 205 by 158 mm) with a rotational mixer was used in this study for combined near-infrared (NIR) and UV-C treatment. A quartz halogen infrared heating lamp (NS-104, 350 mm; NSTECH, South Korea), with a maximum power of 500 W at a 230-V input, was used as a NIR-emitting source. A UV germicidal lamp (G10T5/4P, 357 mm; Sankyo, Japan) with a nominal output power of 16 W (light intensity of 2.62 mW/cm2 at the sample location) was used as a UV-C-emitting source. Since both lamps radiate in all directions, they were placed within aluminum reflectors to focus as much of the radiation as possible uniformly onto the process line and to prevent energy from leaking out of the chamber. After the outputs of the NIR and UV lamps had been stabilized (following 2 min of run time), inoculated samples (250 g) were placed in the treatment chamber for the subsequent inactivation experiments (NIR radiant heating, UV-C irradiation, and simultaneous application of both technologies). All treatments were accompanied by stirring (23 rpm) by means of a rotational mixer in the chamber. For the inactivation mechanism study, 5 ml of cell suspensions kept in glass petri dishes were treated with NIR, UV, and NIR-UV for 3 min under identical conditions. The volume of the cell suspension (5 ml) and the treatment time (3 min) were selected on the basis of the temperatures of red pepper powder during NIR treatment.

Bacterial enumeration.At selected intervals, 25-g treated samples were removed and immediately transferred into sterile stomacher bags (Labplas Inc., Sainte-Julie, Quebec, Canada) containing 225 ml of BPW (detection limit = 10 CFU/g) and homogenized for 2 min with a stomacher (Easy Mix; AES Chemunex, Rennes, France). After homogenization, 1-ml aliquots of sample were 10-fold serially diluted in 9-ml blanks of BPW, and 0.1 ml of sample or diluent was spread-plated onto each selective medium. Xylose lysine desoxycholate agar (XLD; Difco) and sorbitol MacConkey agar (SMAC; Difco) were used as selective media for the enumeration of S. Typhimurium and E. coli O157:H7, respectively. Where low numbers of surviving cells were anticipated, 250 μl of sample was spread-plated onto each of four plates to lower the detection limit. All agar media were incubated at 37°C for 24 h before counting. To confirm the identity of the pathogens, random colonies were selected from the enumeration plates and subjected to biochemical and serological tests. These tests consisted of the Salmonella latex agglutination assay (Oxoid, Ogdensberg, NY) and E. coli O157:H7 latex agglutination assay (RIM; Remel, Lenexa, KS) for S. Typhimurium and E. coli O157:H7, respectively.

Enumeration of injured cells.The overlay (OV) method was used to enumerate injured cells of S. Typhimurium (22). TSA was used as a nonselective medium to repair injured cells. One hundred microliters of appropriate dilutions was spread-plated onto TSA medium in duplicate, and plates were incubated at 37°C for 2 h to allow injured microorganisms to resuscitate (23). Plates were then overlaid with 7 to 8 ml of the selective medium XLD agar. After solidification, plates were further incubated for an additional 22 h at 37°C. Following incubation, presumptive colonies of S. Typhimurium with typical black colonies were enumerated. For enumerating injured cells of E. coli O157:H7, phenol red agar base with 1% sorbitol (SPRAB; Difco) was used (24). After incubation at 37°C for 24 h, typical white colonies characteristic of E. coli O157:H7 were enumerated. Randomly selected isolates from SPRAB plates were subjected to serological confirmation as E. coli O157:H7 (RIM, E. coli O157:H7 latex agglutination test; Remel, Lenexa, KS), because SPRAB is not typically used as a selective agar for enumerating E. coli O157:H7.

Temperature measurement.A fiber optic temperature sensor (FOT-L; FISO Technologies Inc., Quebec, Canada) connected to a signal conditioner (TMI-4; FISO Technologies Inc., Quebec, Canada) was used to measure real-time temperatures in the treatment chamber during combined NIR and UV treatment. The sensor was placed directly on the inner wall surface of the chamber, and the temperature was manually recorded every 5 s. Additionally, in order to measure the core temperature of treated samples precisely, a K-type thermocouple and a data logger (34790A; Agilent Technologies, Palo Alto, CA) were used. The thermocouple probe was directly inserted into the powder bed, and temperatures were recorded at selected treatment times. All experiments were replicated three times.

Transmission electron microscopy analysis.To investigate structural damages in the pathogen cells caused by NIR, UV, and NIR-UV treatments, transmission electron microscopy (TEM) analysis was utilized. Treated S. Typhimurium and E. coli O157:H7 cells in PBS were collected by centrifugation at 4,000 × g for 10 min. The pellet was fixed in modified Karnovsky's fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer) at 4°C for 2 to 4 h. After primary fixation, cells were centrifuged and washed three times with 0.05 M sodium cacodylate buffer. The cells were then postfixed in 1% osmium tetroxide in 0.05 M sodium cacodylate buffer (pH 7.2) at 4°C for 2 h and briefly washed twice with distilled water. The washed cells were prestained in 0.5% uranyl acetate for 30 min at 4°C. The cells were then dehydrated using a graded ethanol series of 30, 50, 70, 80, 90 and three changes of 100% for 10 min each. After dehydration, cells were processed in two changes of 100% propylene oxide (transition material) at room temperature for 15 min each, infiltrated in a 1:1 solution of propylene oxide and Spurr's resin for 2 h, and then placed in Spurr's resin overnight. Infiltrated samples were polymerized at 70°C for 24 h. These specimens were sectioned (slices 70 nm thick) using an ultramicrotome (MT-X; RMC, Tucson, AZ) and then stained with 2% uranyl acetate for 7 min and Reynold's lead citrate for 7 min. The dried sections were examined by TEM (Libra 120; Carl Zeiss, Heidenheim, Germany) and digitally photographed.

Measurement of propidium iodine uptake.The fluorescent dye propidium iodine (PI; Sigma-Aldrich) was used to quantitatively assess membrane damage to pathogen cells induced by each treatment. Treated S. Typhimurium and E. coli O157:H7 cells were diluted in PBS to an optical density at 680 nm (OD680) of approximately 0.4 and then mixed with PI solution to a final concentration of 2.9 μM. After incubation for 10 min, samples were centrifuged at 10,000 × g for 10 min and washed twice in PBS to remove excess dye. The cell pellet was resuspended in PBS, and fluorescence was measured with a spectrofluorophotometer (Spectramax M2e; Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 493 nm and an emission wavelength of 630 nm. Fluorescence values from untreated cells were subtracted from those of treated cells, and the data were normalized against the OD680 of the cell suspensions.

Color and capsaicinoid measurement.A Minolta colorimeter (model CR400; Minolta Co., Osaka, Japan) was used to measure the color changes of treated samples. The color attributes were quantified by the values of L*, a*, and b* and measured at random locations on red pepper powder. L*, a*, and b* values indicate color lightness, redness, and yellowness of the sample, respectively. All measurements were taken in triplicate.

Capsaicinoid measurement was conducted following the method of Attuquayefio and Buckle (25). Each sample of treated red pepper powder (4 g) was mixed with 20 ml of acetonitrile and vortexed for 2 min. One ml of extract was diluted with 9 ml of distilled water and passed into conditioned C18 Sep-pak columns (Waters, MA). A C18 Sep-pak column was conditioned with 5 ml of acetonitrile followed by 5 ml of double-distilled water. The capsaicinoids were then eluted with 4 ml of acetonitrile followed by 1 ml of acetonitrile containing 1% acetic acid. To confirm the quantity of capsaicinoids (total amount of capsaicin and dihydrocapsaicin), high-performance liquid chromatography (HPLC) analysis was performed on a Waters Alliance 2695 separation module with a Waters 996 photodiode array detector (Waters, MA). The wavelength was set at 280 nm, and separation was performed using an INNO C18 column (4.6 mm by 250 mm; particle diameter of 5 μm; Innopia, South Korea) at 35°C. The mobile phase consisted of MeOH-water (70:30, vol/vol) at a flow rate of 1.0 ml/min.

Statistical analysis.All experiments were repeated three times with duplicate samples. Data were analyzed by analysis of variance (ANOVA) and Duncan's multiple range test of a statistical analysis system (SAS Institute, Cary, NC). A P value of <0.05 was used to indicate significant differences.

RESULTS

Inactivation of pathogenic bacteria by NIR-UV simultaneous treatment.Viable-count reductions of S. Typhimurium and E. coli O157:H7 in red pepper powder during NIR radiant heating, UV-C irradiation, and simultaneous application of both technologies are shown in Tables 1 and 2, respectively. The simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log reductions in S. Typhimurium and E. coli O157:H7, respectively. For both pathogens, the sum of NIR and UV inactivation was lower than that obtained by the simultaneous application of both technologies, and the existence of a synergistic effect could be deduced. Furthermore, statistically significant (P < 0.05) differences between the sum of NIR and UV inactivation and inactivation achieved with combination treatment were observed in both S. Typhimurium and E. coli O157:H7 after treatment times of 3 min or more (Tables 1 and 2). In S. Typhimurium, inactivation resulting from the synergistic effect occurred after 3 min of treatment, calculated by subtracting the sum of NIR and UV reductions from those obtained during NIR-UV simultaneous treatment, were 0.48, 1.17, and 1.86 logs at 3, 4, and 5 min of treatment, respectively. In the case of E. coli O157:H7, 0.38-, 0.91-, and 1.31-log reductions of synergism at 3, 4, and 5 min, respectively, were observed.

Recovery of NIR-UV-injured cells.Table 1 and 2 show levels of sublethally injured S. Typhimurium and E. coli O157:H7 cells in red pepper powder following simultaneous NIR-UV treatment. Determining the difference between inactivation of samples subjected to injured-cell recovery methods and those plated directly on selective media revealed the presence of 0.23, 0.14, and 0.32 log units of injured S. Typhimurium cells after 3-, 4-, and 5-min treatments, respectively. In the case of E. coli O157:H7, 0.23 to 0.49 log CFU/g of injured cells was observed after 3 to 5 min of treatment, respectively. Overall, slightly lower reductions of both pathogens were observed by the agar OV method (SPRAB in the case of E. coli O157:H7) than on selective agar. However, statistically significant (P > 0.05) differences between the reduction levels enumerated on the appropriate selective agar (XLD and SMAC) versus the agar for resuscitation (OV-XLD and SPRAB) were not observed after the maximum treatment of 5 min.

Average temperature-time histories of red pepper powder.Average temperatures of the treatment chamber and red pepper powder core during simultaneous NIR and UV treatment are shown in Fig. 1. Differences in temperature (about 10 to 13°C) were detected between the inside of the chamber and the red pepper powder core during 1 to 5 min of treatment. After 3 min of treatment, the treatment chamber and sample core reached ca. 62 and 50°C, respectively. At maximum treatment time (5 min), those temperatures increased to 75 and 62°C, respectively (Fig. 1). Additionally, the heating rate of single NIR treatment was not different from that of the NIR-UV simultaneous treatment (data not shown).

Microscopic evaluation of damages.Selected TEM images of ultrastructural changes in S. Typhimurium and E. coli O157:H7 cells induced by NIR, UV, and NIR-UV treatments are shown in Fig. 2 and 3, respectively. Microscopic analyses at the cellular level verified that there was cytoplasmic and membrane structural damage during NIR heating (Fig. 2C and 3C) and simultaneous NIR-UV treatment (Fig. 2D and 3D). More specifically, for both pathogens, cytoplasmic shrinkage and aggregation were observed in both NIR- and NIR-UV-treated cells, in contrast to untreated and UV-treated cells. Furthermore, NIR-UV-treated cells experienced significant cell wall damage, leading to a leakage of cellular contents from the cytoplasm. In the case of UV-treated cells, morphological changes as well as collapse of internal cellular structures were not observed compared to control cells (Fig. 2B and 3B).

Determination of membrane damage by PI uptake.As a further quantitative test of membrane damage, NIR-, UV-, and NIR-UV-treated cells were stained with the fluorescent dye PI, which is excluded from cells with intact membranes. Table 3 shows the PI uptake values of S. Typhimurium and E. coli O157:H7 after each treatment. The overall pattern of results for E. coli O157:H7 was similar to that of S. Typhimurium. Based on PI uptake values, there was no significant (P > 0.05) damage to cellular membranes of either pathogen following UV treatment. The degree of PI uptake in NIR- and NIR-UV treated cells was much greater than that in UV-treated cells. Among them, the cells subjected to NIR-UV treatments showed significantly (P < 0.05) higher PI uptake values than did cells subjected to the other treatments.

Effect of NIR-UV simultaneous treatment on product quality.Color values of red pepper powder after NIR-UV combined treatment are presented in Table 4. L*, a*, and b* values of NIR-UV treated (5 min) red pepper powder were not significantly (P > 0.05) different from those of untreated samples. Natural red color is a major attribute for determining the commercial quality of red pepper powder. A red color parameter was expressed as the product of L* (lightness) and a* (redness); values of >500, 500 to 300, and 300 were rated as red, medium red, and dark red, respectively (26). The red color parameters for all treatments stayed within acceptable levels (L* × a* ∼ 700). Since the capsaicinoids, primarily capsaicin and dihydrocapsaicin, present in red pepper are responsible for its typical pungency and flavor, retention of these constituents during a given process is very important for ensuring acceptability of the commodity. Table 5 shows that NIR-UV simultaneous treatment for 5 min did not change the content of capsaicin and dihydrocapsaicin of red pepper powder significantly (P > 0.05). Thus, simultaneous application of NIR and UV treatment for 5 min did not affect the quality of red pepper powder product.

DISCUSSION

After single treatment with NIR heating for 5 min, there was inactivation of 1.45 and 1.42 log in S. Typhimurium and E. coli O157:H7, respectively. To achieve >3-log reductions, based on the calculated parameters of the Weibull model (R2 = 0.96), 6.5 min and 7.2 min were needed for S. Typhimurium and E. coli O157:H7, respectively. However, darkening was visually observed on red pepper powder treated with NIR for a slightly excessive time (over 6.5 min). Moreover, treatment of red pepper powder with UV-C, where approximately 0.04-log reductions were observed in each pathogen after 5 min of exposure, was less effective than NIR treatment. A review of the literature shows that there have been only a few studies relevant to the effect of UV-C irradiation on spices. Erdoǧdu and Ekiz (12) reported that the use of UV-C irradiation alone on cumin seeds for 60 min reduced total aerobic bacteria by about 0.6 log unit, and extending the UV-C application up to 120 min did not offer any additional reduction. As these results indicate, the effects of UV-C and NIR treatments alone were not effective in reducing the number of pathogens found in agricultural spices while maintaining product quality.

Hamanaka et al. (21) investigated sequential UV-C and NIR treatment for surface decontamination of fresh fig fruit and reported that IR heating accelerated the cell-inactivating efficacy of UV-C irradiation. However, separate 30-s treatments of IR heating and UV irradiation reduced fungal counts by about 1 and 2.5 logs, respectively, in comparison with the control, while sequential treatment of IR heating and UV irradiation reduced fungal populations by 3 logs. Thus, sequential application of IR heating followed by UV treatment showed an additive effect that was not significantly different from the sum of IR and UV inactivation. This result was consistent with another report describing Salmonella inactivation by the combination of UV-C light with mild heat (27). Gayán et al. (27) reported that the sequential combination of heat (55°C) followed by UV treatment showed an additive effect and also noted that a synergistic effect was observed when both technologies were applied simultaneously. Therefore, a simultaneous combination of NIR heating and UV-C irradiation was included in our study, and the required levels of protection (≥3 log reduction) could be obtained based on synergism while retaining organoleptic attributes of red pepper powder, such as color and pungency.

Even though NIR-UV treatment was highly effective, the significance of sublethally injured pathogens in food samples should not be ignored. Injured cells are potentially as dangerous as their normal counterparts because they can resuscitate and become functionally normal under suitable conditions (28). In this study, the occurrence of sublethally injured pathogens in powdered red pepper was assessed by plating on selective agars with and without a resuscitation step. There were no significant (P > 0.05) differences in levels of cells enumerated on XLD and OV-XLD and those on SMAC and SPRAB after the maximum treatment of 5 min (Tables 1 and 2). This suggests that simultaneous NIR-UV treatment effectively inactivated S. Typhimurium and E. coli O157:H7 in powered red pepper without causing appreciable injury to bacterial cells.

The underlying inactivation mechanisms of the combination of UV-C irradiation and NIR heating are not well understood. In previous studies, Sawai et al. (29) investigated the inactivation mechanism of E. coli treated with IR radiation in phosphate-buffered saline. They reported that IR irradiation damaged RNA polymerase and ribosomes before damaging DNA and cell membranes. Additionally, RNA, protein, and cell walls showed greater vulnerability to IR heating than conductive heating. Gayán et al. (27) reported that the number of envelope-injured cells was higher after combined UV and heat processing than after heating alone, and this difference was more pronounced in outer membrane than in cytoplasmic membrane damage. Their results indicate that the mechanism of the synergistic effect of UV irradiation and heat is relevant to a sensitization of cell envelopes or the inability of cells to repair these structures.

To clarify the mechanism of the synergistic lethal effect of NIR-UV combined treatment, membrane damage to S. Typhimurium and E. coli O157:H7 cells caused by NIR, UV, and NIR-UV simultaneous treatment was evaluated by qualitative (TEM analysis) and quantitative (PI uptake test) methods. For this purpose, a 3-min treatment time was chosen, since the significant (P < 0.05) synergistic effects were detected at ≥3 min of treatment in both S. Typhimurium and E. coli O157:H7 (Table 1 and 2). The NIR-UV combined treatment significantly damaged cell envelopes of both pathogens, as detected in both sets of TEM images (Fig. 2 and 3) and PI uptake values (Table 3). Fluorescent stains that bind to intracellular components are useful in determining the viability or the physiological status of microorganisms. In particular, PI is a nucleotide-binding probe, which enters only cells with damaged membranes (30). Quantitative results of cell membrane damage measured by PI uptake were consistent with qualitative observations obtained by TEM analysis (Fig. 2 and 3) and logarithmic inactivation results obtained from each selective medium (Table 1 and 2). The synergism of NIR-UV simultaneous treatment evident in PI uptake values was also observed in log reduction data of each pathogen. The PI uptake values of simultaneous NIR-UV application were higher than total values reached by separate NIR and UV treatments for both S. Typhimurium and E. coli O157:H7 (Table 3). As a result, we confirmed that damage to the cell envelope was the main factor related to the synergistic lethal effect of combined NIR and UV treatment. Although it is well established that UV irradiation inactivates bacteria by damaging their nucleic acids, it has also been suggested that photons can interact with components of cell envelopes, including the cell wall and membrane (29). However, in our mechanism study, TEM images and PI uptake values of UV-treated cells were not significantly different from those of untreated cells.

In studying the effect of excessive thermal treatment on the quality properties of spices, Almela et al. (31) noted that high temperature promotes color degradation of spices, as paprika became darker and the red color became less intense. Rico et al. (2) also reported that steaming treatment of red pepper powder led to quality degradation, with significant loss of color and flavor. Thus, it is important that spices be heated no longer than required, in order to prevent losses in flavor and color components. As shown in our study, simultaneous NIR-UV treatments did not change color values or capsaicin and dihydrocapsaicin content of red pepper powder significantly (P > 0.05), due to the shorter NIR processing time (Tables 4 and 5). Also, in the present study, because a rotational mixer was used simultaneously with combined NIR-UV treatment, excessive heating on one side of the powder mass was prevented. Furthermore, since UV-C radiation as well as NIR thermal energy is primarily absorbed on solid food surfaces and has very limited penetration capability, simultaneous mixing could increase the contact area of radiation on powder particles. For these reasons, rotational mixing should be incorporated on an industrial scale when powdered spices are being decontaminated.

In conclusion, performing simultaneous UV-C irradiation with NIR heating to temperatures lower than those used for inactivation was found to be suitable for reducing microbial contamination of red pepper powder without affecting product quality. The NIR-UV combination has some advantages not only regarding the germicidal effect but also in terms of simplified handling, environmental preservation, and reduced costs through lower inputs of energy. These factors suggest that this decontaminating procedure can be applied as an alternative to other interventions, such as fumigation and superheated steam. In addition, data obtained from this novel system will be useful in designing apparatus for NIR and UV-C combined bactericidal processing for various kinds of powdered/granulated food ingredients.

ACKNOWLEDGMENTS

This research was supported by R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported by the Public Welfare & Safety research program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012M3A2A1051679).

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